Current management of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) relies solely on symptom management to improve quality of life but does not address the underlying mechanisms of disease onset and progression. In an effort to provide insight into the key biological targets involved CFS/ME presentation, the main objective of this research proposal is to identify novel biomarkers and therapeutic targets of CFS/ME and provide insight into disease onset and progression. This research proposal aims to use Peripheral Blood Mononuclear Cells (PBMC) from patients recruited for our recently NIH funded research. These PBMC have been isolated from CFS/ME patients and matched healthy controls at three time points - before exercise challenge, at the peak of effort (VO2 max) and four hours after the peak effort. Specific Aims of this proposal include: Specific Aim 1. Identify metabolic pathways affected at CFS/ME and possible regulatory RNAs. Based on our previous findings, our hypothesis is that unknown transcripts and alternative splicing events regulate changes in expression of genes responsible for inflammatory response, immune system processes, leukocyte migration, and regulation of cell development. This is leading to changes in CFS/ME disease activity. We aim to expand our research efforts, by using an RNA-seq approach to identify the abundance of transcripts, discover new splicing events, and presence of novel transcripts. This will allow identifying biomarkers as a result of differentially expressed transcripts and alternative splicing and non-coding RNAs to establish a set of CFS/ME specific candidate transcripts. We will then validate them using NanoString Technologies' nCounter system and create a panel of transcripts to potentially be used in the diagnosis and management of CFS/ME. Specific Aim 2. To investigate possible mechanisms of transcriptional regulation in CFS/ME. To better understand causes of changes in gene expression in patients with CFS/ME, we aim to identify changes in copy number variation (CNV) and genomic DNA methylation. We will use Agilent SurePrint G3 Human Genome CGH+SNP microarrays to determine CNV in CFS/ME and identify a set of CFS/ME-specific candidate CNVs. We will use Illumina Infinium Human Methylation450 BeadChip microarrays as well as real-time probe-based PCR to evaluate differences in the methylation patterns between CFS/ME patients and healthy controls. We will combine the results of RNA-seq, CNV and methylation assays and research correlation to determine possible mechanisms of transcriptional regulation involved in CFS/ME, which will lead to the better targeted therapeutic intervention.